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. 2015 Nov 6;11(11):e1005255. doi: 10.1371/journal.ppat.1005255

Fig 2. KSHV infection increases expression from the miR-17-92 cluster promoter via vFLIP and vCyclin.

Fig 2

(A) Uninfected SLK cells and SLK cells latently infected with KSHV were electroporated with reporter vector (pGL3-PmiR-17-92) containing the promoter of the miR-17-92 cluster upstream of luciferase gene [19]. The firefly luciferase activity was measured then set at 1 for uninfected SLK cells. Error bars show standard deviation for triplicate experiments. (B) Co-transfection of pGL3-PmiR-17-92 with plasmids expressing LANA or the KSHV miRNA cluster. pGL3-PmiR-17-92 was co-transfected with empty vector or with pcDNA3/LANA or pcDNA3.1/cluster into SLK cells using Mirus293IT transfection reagent. Firefly and Renilla luciferase activities were measured, and firefly luciferase activity was normalized to control Renilla luciferase activity. Activity was set at 1 for cells transfected with empty vector. (C) Confirmation of expression of V5-tagged vFLIP and vCyclin by Western blotting. Extracts of cells transfected with pLenti6/vFLIP or pLenti6/vCyc were probed with anti-V5 mAb. D) Co-transfection of pGL3-PmiR-17-92 with plasmids expressing vFLIP or vCyclin. pLenti6/vFLIP or pLenti6/vCyc were co-transfected with reporter vector into SLK cells and firefly luciferase activity measured and normalized as above.